Fig. 1
Primers and TaqMan probes for qPCR of pathogenic Leptospira spp_
| Target gene | Nucleotide sequence | Reference |
|---|---|---|
| LipL32(1) | ||
| Forward | 5′-AAG CAT TAC CGC TTG TGG TG-3′ | 36 |
| Reverse | 5′-GAA CTC CCA TTT CAG CGA TT-3′ | |
| TaqMan probe | FAM/ZEN-5’-AA AGC CAG GAC AAG CGC CG-3’-IBFQ | |
| LipL32(2) | ||
| Forward | 5′-CGG GAG GCA GCA GTT AAG AAT-3′ | 41 |
| Reverse | 5′-ACG TAT GGT GCA AGC GTT GTT-3′ | |
| TaqMan probe | FAM/ZEN-5′-GCA ATG TGA TGA TGG TAC CTG CCT-3′-IBFQ | |
Threshold cycle (Ct) values of fivefold serial dilution of Leptospira genomic DNA in a 20 μL reaction volume
| Dilution factor | Mean DNA copy number/μL | log DNA copies | Mean Ct value |
|---|---|---|---|
| Standard control with DNA concentration of 51.85 ng/μL (primary stock) | 10,382,320 | 7.01629441 | 12.47 |
| 1:5 | 2,076,464 | 6.317324406 | 14.15 |
| 1:25 | 415,292.8 | 5.618354402 | 17.66 |
| 1:125 | 83,058.56 | 4.919384397 | 18.96 |
| 1:625 | 16,611.712 | 4.220414393 | 21.05 |
| 1:3,125 | 3,322.3424 | 3.521444389 | 23.36 |
| 1:15,625 | 664.46848 | 2.822474384 | 25.79 |
| 1:78,125 | 132.893696 | 2.12350438 | 27.43 |
| 1:390,625 | 26.5787392 | 1.424534376 | 29.67 |
| 1:1,953,125 | 5.31574784 | 0.725564371 | 32.00 |
Comparison of Ct values between a qPCR assay with the LipL32(1) primers and one with the LipL32(2) primers
| Group | DNA concentration (ng/μL) | LipL32(1) Ct value | LipL32(2) Ct value |
|---|---|---|---|
| Positive control | |||
| L. borgpetersenii serovar Ballum strain Mus 127 | 26.4 | 15.33 | 32.08 |
| L. borgpetersenii serovar Hardjobovis strain 117123 | 20.6 | 17.41 | 33.36 |
| L. borgpetersenii serovar Javanica* | 8.2 | 16.22 | 36.14 |
| L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46 | 41.0 | 14.35 | 33.96 |
| L. borgpetersenii serovar Tarassovi strain Perepelitsin | 16.1 | 20.54 | 36.39 |
| L. interrogans serovar Australis* | 9.9–11.9 | 15.43–15.58 | 33.78–34.38 |
| L. interrogans serovar Australis strain Ballico | 11.8 | 29.67 | N |
| L. interrogans serovar Autumnalis strain Akiyami A | 32.6 | 23.56 | N |
| L. interrogans serovar Bataviae* | 5.3–20.1 | 13.91–16.02 | 34.11–35.91 |
| L. interrogans serovar Bataviae strain Swart | 15.8 | 17.97 | 34.29 |
| L. interrogans serovar Canicola strain Hond Utrecht IV | 12.7 | 14.99 | 34.08 |
| L. interrogans serovar Copenhageni strain M20 | 2.5 | 17.95 | 34.44 |
| L. interrogans serovar Djasiman strain Djasiman | 11.5 | 16.20 | 35.43 |
| L. interrogans serovar Hebdomadis strain Hebdomadis | 28.4 | 16.77 | 33.91 |
| L. interrogans serovar Icterohaemorrhagiae strain RGA | 16.8 | 14.3 | 32.81 |
| L. interrogans serovar Lai strain Lai | 12.8 | 19.22 | 36.19 |
| L. interrogans serovar Pomona strain Pomona | 7.2 | 18.05 | 35.44 |
| L. interrogans serovar Pyrogenes strain Salinem | 43.3 | 21.40 | N |
| L. kirschneri serovar Cynopteri strain 3522C | 29.7 | 14.44 | 33.60 |
| L. kirschneri serovar Grippotyphosa strain Moskva V | 42.3 | 13.04 | 31.79 |
| L. kmetyi serovar Malaysia strain Bejo-ISO9 | 36.1 | 18.78 | 33.34 |
| L. weilii serovar Celledoni strain Celledoni | 13.0 | 24.15 | 40.52 |
| Non-target negative control | |||
| Bacillus spp. | 9.9 | N | N |
| Escherichia coli | 16.0 | N | N |
| L. biflexa serovar Patoc strain Patoc I | 42.9 | N | N |
| L. selangorensis** | 8.7-14.3 | N | N |
| L. wolffii** | 6.7-19.3 | N | N |
| Pseudomonas aeruginosa | 84.9 | N | N |
| Salmonella spp. | 41.1 | N | N |
| Staphylococcus aureus | 14.4 | N | N |
| Streptococcus spp. | 4.2 | N | N |
| No-target negative control | |||
| RNase-free water | 0 | N | N |
List of bacteria used in the qPCR assay
| Bacterium (n = 44) | Number tested (n) | Source |
|---|---|---|
| Pathogenic group | ||
| L. borgpetersenii serovar Ballum strain Mus 127 | 1 | FVM, UPM |
| L. borgpetersenii serovar Hardjobovis strain 117123 | 1 | FVM, UPM |
| L. borgpetersenii serovar Javanica | 1 | Dog |
| L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46 | 1 | FVM, UPM |
| L. borgpetersenii serovar Tarassovi strain Perepelitsin | 1 | FVM, UPM |
| L. interrogans serovar Australis | 2 | Dog |
| L. interrogans serovar Australis strain Ballico | 1 | FVM, UPM |
| L. interrogans serovar Autumnalis strain Akiyami A | 1 | FVM, UPM |
| L. interrogans serovar Bataviae | 8 | Dog |
| L. interrogans serovar Bataviae strain Swart | 1 | FVM, UPM |
| L. interrogans serovar Canicola strain Hond Utrecht IV | 1 | FVM, UPM |
| L. interrogans serovar Copenhageni strain M20 | 1 | FVM, UPM |
| L. interrogans serovar Djasiman strain Djasiman | 1 | FVM, UPM |
| L. interrogans serovar Hebdomadis strain Hebdomadis | 1 | FVM, UPM |
| L. interrogans serovar Icterohaemorrhagiae strain RGA | 1 | FVM, UPM |
| L. interrogans serovar Lai strain Lai | 1 | FVM, UPM |
| L. interrogans serovar Pomona strain Pomona | 1 | FVM, UPM |
| L. interrogans serovar Pyrogenes strain Salinem | 1 | FVM, UPM |
| L. kirschneri serovar Cynopteri strain 3522C | 1 | FVM, UPM |
| L. kirschneri serovar Grippotyphosa strain Moskva V | 1 | FVM, UPM |
| L. kmetyi serovar Malaysia strain Bejo-ISO9 | 1 | FVM, UPM |
| L. weilii serovar Celledoni strain Celledoni | 1 | FVM, UPM |
| Intermediate group | ||
| L. selangorensis | 3 | Environmental |
| L. wolffii | 5 | Environmental |
| Saprophytic group | ||
| L. biflexa serovar Patoc strain Patoc I | 1 | FVM, UPM |
| Other bacteria | ||
| Bacillus spp. (Gram positive) | 1 | Clinical |
| Escherichia coli (Gram negative) | 1 | Clinical |
| Pseudomonas aeruginosa (Gram negative) | 1 | Clinical |
| Salmonella spp. (Gram negative) | 1 | Clinical |
| Staphylococcus aureus (Gram positive) | 1 | Clinical |
| Streptococcus spp. (Gram positive) | 1 | Clinical |